SHORT PAPER Random Loss of X Chromosome at Male Determination in an Aphid, Sitobion Near Fragariae, Detected Using an X-Linked Polymorphic Microsatellite Marker

Genet. Res., Camb.(1997), 69, pp. 233–236. With 1 ﬁgure. Printed in the United Kingdom # 1997 Cambridge University Press 233

SHORT PAPER Random loss of X chromosome at male determination in an aphid, Sitobion near fragariae, detected using an X-linked polymorphic microsatellite marker

ALEXANDRA C.C. WILSON*, PAUL SUNNUCKS  DINAH F. HALES School of Biological Sciences, Macquarie Uniersity NSW 2109, Australia (Receied 28 October 1996 and in reised form 7 February 1997)

Summary This paper reports the ﬁrst direct molecular evidence that X chromosome loss during determination of male aphids (XO) is random. Clonal and sexual females, and males, of the species Sitobion near fragariae were screened using three polymorphic microsatellite loci. Two loci, Sm10 and Sm17, showed the same heterozygous genotypes in all three aphid morphs. The third, Sm11, was heterozygous for the same two alleles in clonal and sexual females, but of the 25 males screened 11 showed the ‘160’ allele and 14 showed the ‘156’ allele. This result indicates X- linkage of locus Sm11, with random loss of the X chromosome during the formation of male embryos. The possible implications of this result are discussed with respect to aphid sex determination, recombination and chromosome evolution.

The Coccoidea (scale insects, mealybugs) constitute 1. Introduction the sister group to the aphids within the Suborder Reproduction in Aphididae encompasses a large range Sternorrhyncha of the Order Hemiptera (Moran et of strategies, including obligate apomictic partheno- al., 1994). There is a wide range of bizarre cytological genesis, and a facultative yearly sexual generation events in diﬀerent coccoid families, often including a with one to many intervening parthenogenetic genera- form of imprinting of the paternal chromosomes such tions (for review see Hales et al., 1997). Sex de- that they are inactive. For example, in lecanoid scale termination in most aphids is XX\XO. All aphid insects, the paternal chromosomes become hetero- zygotes are originally XX female, but some become chromatic in male embryos at the blastoderm stage, male on receiving only one X chromosome during and are ultimately eliminated during spermatogenesis division of an oocyte in a parthenogenetic female at the second meiotic division, so that males pass on (Orlando, 1974; Blackman & Hales, 1986). Multiple only those chromosomes received from their mothers. X chromosome systems are also known in aphids In diaspidid scale insects, the whole paternal set of (Blackman, 1995). There are still many unanswered chromosomes is completely eliminated in male em- questions about chromosomal behaviour during male bryos at late cleavage (White, 1973). meiosis and male determination in aphids (see above Crema (1981) found an approximately equal ratio references). Male meiosis in aphids is peculiar in that of aborted eggs to viable male eggs in Megoura iciae only those autosomes that associate with an X and suggested that it was attributable to a recessive chromosome survive and the set of autosomes lacking lethal gene on one of the X chromosomes. Further, an X chromosome receives little cytoplasm and does Smith & MacKay (1989) inferred that the ratio of 1:1 not form a viable spermatocyte (for review of early winged to wingless phenotype in males of Acyrtho- work see Blackman, 1987). Factors determining which siphon pisum resulted from a sex-linked gene. Their autosomes become associated with an X chromosome data suggested that X chromosome elimination was are not known. Even during parthenogenetic re- random with respect to chromosomal identity. How- production, in which oogenesis is essentially mitotic, ever, wing phenotype is a highly labile feature in observations of chromosome behaviour suggest the aphids, responding to numerous environmental vari- possibility of X chromosome recombination (Black- ables acting in late embryonic or early larval de- man & Hales, 1986; Blackman & Spence, 1996), but velopment (reviewed by Hille Ris Lambers, 1966; this has not been tested with genetic data. Lees, 1966; Hales, 1976; Kawada, 1987), and it is thus desirable to test for equality of elimination of each X * Corresponding author. Tel: (612) 9850 8223. Fax: (612) 9850 8245. e-mail: awilson!rna.bio.mq.edu.au. chromosome using appropriate molecular markers. Downloaded from https://www.cambridge.org/core. IP address: 170.106.35.93, on 30 Sep 2021 at 06:28:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672397002747 A. C. C. Wilson et al. 234

It cannot be assumed that there is an equal 2. Materials and methods probability of elimination of either of the X chromosomes at male determination in aphids, nor of the A single clone (clone 17) of Sitobion near fragariae elimination of either set of autosomes at male meiosis. originating from Tasmania was raised on barley We have previously attempted to address the former seedlings in pots in a temperature-controlled room at issue using allozymes, but did not find a polymorphic 15 mC and an 8 h light\16 h dark photoperiod. Under sex-linked marker in any species studied (D. F. Hales, these conditions parthenogenetic winged and wingless unpublished data). The discovery of an X-linked females, sexual females and males were produced. microsatellite has provided us with a molecular marker Sexual females and males were stored in 70% ethanol. of suitable resolution to study the probability of (Under the same conditions, clones of S. miscanthi, elimination of X chromosomes in aphids at male another aphid of grasses and cereals in Australia, did determination. not produce sexual forms.) Microsatellites are co-dominant, single-locus DNA from individual aphids was extracted using a markers which exhibit a large range of genetic diversity ‘salting-out’ protocol (Sunnucks & Hales, 1996) and and are relatively neutral to selection (Bruford & dissolved in 20 µl of TE Buffer (1 m EDTA, 10m Wayne, 1993; Hughes & Queller, 1993; Estoup et al., Tris base pH 7n5). Microsatellites were cloned and 1995; England et al., 1996). They provide powerful developed from Sitobion miscanthi as described in tools for linkage and population studies. X-linked Sunnucks et al.(1996). The sequence of primers for microsatellites have previously been reported in other loci Sm10 and Sm17 will be published elsewhere, but insects including Drosophila melanogaster (Goldstein in the meantime may be requested from the authors. & Clark, 1995; England et al., 1996) and a mosquito, The sequences of primers to amplify locus Sm11 were Anopheles gambiae (Lanzaro et al., 1995). (5h to 3h): Sm11F aac cct acg ggt aac gcc (kTm l Sitobion near fragariae is a grass aphid thought to 59n8 mC) and Sm11R ggt acc cct atg tta tta cgc g have arrived in Australia within the last 50 years, and (kTm l 59n7 mC). is now common in south-eastern Australia. It is Microsatellite polymerase chain reaction (PCR) morphologically similar, but not identical, to S. was carried out in 10 µl reaction volumes with 0n1 µl #+ fragariae (Walker) from the Holarctic region. Mol- of Taq polymerase (0n5 units, Promega), Mg -free ecular evidence also supports a close relationship of reaction buffer, 2 m MgCl#, 200 µ dGTP, dCTP this species with S. fragariae (Sunnucks & Hales, and dTTP, 20 µ dATP, 5–10 pmoles of primers, 5– 1996). 50 ng DNA (typically 1 µl as prepared above) and $$ Males, but not sexual females, of S. near fragariae 0n05 µlof[αP]dATP (10 mCi\ml; Bresatec). The have been reported in field collections in Australia (M. PCR cycling conditions for locus Sm11 were: 94 mC for Carver, personal communication). These aphids under 3 min, four cycles of ‘touch-down’ (annealing first at field conditions appear to be behaving as obligate 55 mC for 30 s and decreasing for the next three cycles parthenogens: we have used microsatellites to dem- (53 mC, 51 mC, 49 mC), extending at 72 mC for 45 s, onstrate the absence of genetic recombination in over denaturing at 94 mC for 15 s) and then cycling for 29 150 Australian S. near fragariae from diverse col- cycles, annealing at 47 mC, with a final extension at lections (Sunnucks et al., 1996, and unpublished 72 mC for 2 min. For Sm10 and Sm17, all annealing data). However, under laboratory conditions we have temperatures were increased by 8 mC. Four microlitres induced a clone of S. near fragariae to produce males of each reaction were run on a 6% acrylamide and sexual females (present study). sequencing gel using the positive control M13 DNA The cloning of microsatellites has given us the tools of the Sequenase version 2.0 kit (United States to investigate X-linkage and, further, we have analysed Biochemicals) as a marker ladder for the precise sizing sufficient males to determine whether the elimination of alleles. Reference DNA can be obtained from the of the X chromosome is random at male deter- authors where confirmation of allelic length identity is mination. We are not aware of previous reports of X- necessary. linked microsatellites (or indeed of any highly variable Lengths of chromosomes in air-dried preparations X-linked single loci) in aphids. The relevance of our (Hales & Lardner, 1988) from Sitobion clones were study to recently published work concerning rDNA measured from photographic prints using vernier (Blackman & Spence, 1996), and the observations of calipers, and X chromosome length as a percentage of ‘aborted’ embryos during the transition to male haploid genome length in each chromosome set was production (Crema, 1981; Searle & Mittler, 1981)is calculated (n l 8; 3 S. near fragariae and 5 S. discussed. We show that microsatellite markers should miscanthi). be valuable in elucidation of cytogenetic observations The G-test for goodness of fit (Sokal & Rohlf, 1995) of aphid male meiosis, and of possible recombination was used to test the observed allelic frequencies in between X chromosomes during parthenogenetic and males at locus Sm11 against equal hemizygous sexual reproduction. frequencies predicted if each maternal X chromosome has a 50% chance of being observed in a given male offspring.

Downloaded from https://www.cambridge.org/core. IP address: 170.106.35.93, on 30 Sep 2021 at 06:28:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672397002747 X-linked microsatellite in aphids 235

females males 3. Results CS A Six sexual females of S. near fragariae clone 17 screened using the microsatellite loci all had, as expected, genotypes identical to those found in 121 parthenogenetic females of the same species screened 160 during a previous study (Sunnucks et al., 1996) (Sm10 156 alleles 157\161;Sm11 156\160; Sm17 174\178). Twenty-five males of Clone 17 showed genotypes at loci Sm10 and Sm 17 identical to those of the parthenogenetic and sexual females. At locus Sm11 each male showed either allele 160 or allele 156 (Fig. 1). Of the 25 males screened, 11 had only allele 160 Fig. 1. Autoradiograph showing phenotypes at locus and 14 had only allele 156 (no significant deviation Sm11 in clonal (‘C’), sexual (‘S’) females and males. 1 from a 1:1 ratio of alleles 160 and 156; G 0 362, Allele sizes are marked in base pairs; M 3 DNA in lane l n A was the size marker (‘A’). P " 0n5). The X chromosome in Sitobion aphids represents 28% (p3%, nl8) of the total length of the haploid Recently, Blackman & Spence (1996) observed that genome. aphids which have lost the sexual phase of their annual cycle frequently have multiple rDNA copies 4. Discussion concentrated on one X chromosome; they suggested that such a situation could result from recombination The present data indicate that microsatellite locus between the X chromosomes during parthenogenesis. Sm11 is sex-linked in S. near fragariae, and that, Further, they investigated the ability of these aphids during the formation of an oocyte that will give rise to to produce males. They found that the loss of rDNA a male, the two X chromosomes have an equal chance arrays from one of the X chromosomes did not of being retained. All parthenogenetic and sexual prevent the formation of males in a clone of females (XX) were found to be heterozygous for Acyrthosiphon pisum. However, if an X chromosome alleles 156\160 of locus Sm11, and all males (XO) passes into males independently of its ancestry, 50% were found to carry only a single allele, 156 or 160, in of male embryos will be deficient in rDNA and should proportions that did not differ significantly from therefore be non-viable. Male-producing aphids are equality. It is unlikely that this result could be due to frequently observed to carry ‘aborted’ embryos recombination of autosomes during the formation of amongst their developing embryos (reviewed in males, because we would then expect to find some Blackman, 1987). Crema (1981; cited in Blackman, heterozygous males and did not. Also there is no 1987) observed in a strain of Megoura iciae that the credible cytological evidence that such recombination numbers of aborted eggs and viable male eggs were in occurs (references in Hales et al., 1997). equal proportion. Given the 1:1 association of aborted We found no evidence of imprinting of chromo- and viable male embryos we propose that these somes in aphids at male determination, inasmuch as aborted embryos may have inherited an X chromo- neither the maternal nor the paternal X chromosome some with insufficient rDNA. Initial development of was selectively eliminated. In contrast, in the closely these embryos may occur by use of maternal ribo- related scale insects, paternal chromosomes are inacti- somes: aphid ovaries are telotrophic, allowing trans- vated or eliminated in early embryonic development port of maternal materials down a nutritive cord to of males: a clear indication of imprinting. This embryos in the second or third position from the difference in imprinting may be related to differences germarium. in the reproductive modes of the two groups. Sexually Being XO, aphid males provide the opportunity to reproducing scale insects have males in every gen- investigate chromosomal haplotypes (linkages) at eration, while many aphids, including S. near frag- multiple loci. We found one X-linked microsatellite ariae, may have many parthenogenetic generations out of three; since X chromosomes represent 28% of between the zygote and the next production of males the genome in our sample of Sitobion aphids, it seems and sexual females. Possibly imprinting does not likely that more X-linked microsatellites can be found efficiently penetrate through many asexual genera- at about the same rate. It will then be possible to test tions. However, random elimination of X chromo- the important and outstanding issues of X chromosomes at sex determination in aphids does not preclude some recombination in sexual and asexual aphids (see some form of expressional imprinting of at least some Blackman, 1987; and review in Hales et al., 1997). genes on X chromosomes or autosomes. The possibility of paternal imprinting having effects several We thank the two anonymous reviewers and Dick Frankham for their comments on the manuscript. This work was generations later could provide an interesting field for supported by an Australian Research Council Small Grant further research. to D.F.H., by Macquarie University Funds, and by Key

Centre for Biodiversity and Bioresources funding to Hales, D. F., Tomiuk, J., Wo$ hrmann, K. & Sunnucks, P. A.C.C.W. This paper is Key Centre for Biodiversity and (1997). Evolutionary and genetic aspects of aphid biology: Bioresources Publication No. 225. a review. European Journal of Entomology 94, 1–55. Hille Ris Lambers, D. (1966). Polymorphism in Aphididae. References Annual Reiew of Entomology 11, 47–78. Hughes, C. R. & Queller, D. C. (1993). Detection of Blackman, R. L. (1987). Reproduction, cytogenetics and polymorphic microsatellite loci in a species with little development. In Aphids: Their Biology, Natural Enemies allozyme polymorphism. Molecular Ecology 2, 131–138. and Control, vol. 2A (ed. A. K. Minks & P. Harrewijn), Kawada, K. (1987). Polymorphism and morph determi- pp. 163–195. Amsterdam: Elsevier. nation. In Aphids: Their Biology, Natural Enemies and Blackman, R. L. (1995). Sex determination in insects. In Control, vol. 2A (ed. A. K. Minks & P. Harrewijn), pp. Insect Reproduction (ed. S. R. Leather & J. Hardie), pp. 255–266. Amsterdam: Elsevier. 57–94. Boca Raton, Florida: CRC Press. Lanzaro, G. C., Zheng, L., Toure, Y. T., Traore, S. F., Blackman, R. L. & Hales, D. F. (1986). Behaviour of the X Kafatos, F. C., & Vernick, K. D. (1995). Microsatellite chromosome during growth and maturation of par- DNA and isozyme variability in a West African popu- thenogenetic eggs of Amphorophora tuberculata (Homop- lation of Anopheles gambiae. Insect Molecular Biology 4, tera, Aphididae), in relation to sex determination. 105–112. Chromosoma 94, 59–64. Lees, A. D. (1966). The control of polymorphism in aphids. Blackman, R. L. & Spence, J. M. (1996). Ribosomal DNA Adances in Insect Physiology 3, 207–277. is frequently concentrated on only one X chromosome in Moran, N. A., Baumann, P. & von Dohlen, C. (1994). Use permanently apomictic aphids, but this does not inhibit of DNA sequences to reconstruct the history of the male determination. Chromosome Research 4,314–320. association between members of the Sternorrhyncha Bruford, M. W. & Wayne, R. K. (1993). Microsatellites and (Homoptera) and their bacterial endosymbionts. Euro- their application to population genetic studies. Current pean Journal of Entomology 91, 79–83. Opinion in Genetics and Deelopment 3, 939–943. Orlando, E. (1974). Sex determination in Megoura iciae Crema, R. (1981). Lethal genes in a parthenogenetic strain Buckton (Homoptera, Aphididae). Monitore Zoologica of Megoura iciae. Bolletino di Zoologia 48, 37–89. Italiano 8,61–70. England, P. R., Briscoe, D. A. & Frankham, R. (1996). Searle, J. B. & Mittler, T. E. (1981). Embryogenesis and the Microsatellite polymorphisms in a wild population of production of males by apterous viviparae of the green Drosophila melanogaster. Genetical Research 67, 285–290. peach aphid Myzus persicae in relation to photoperiod. Estoup, A., Garnery, L., Solignac, M. & Cornuet, J.-M. Journal of Insect Physiology 27, 145–153. (1995). Microsatellite variation in honey bee Apis mellifera Smith, M. A. H. & MacKay, P. A. (1989). Genetic variation (L.) populations: hierarchical genetic structure and test of in male alary dimorphism in populations of pea aphid, the inﬁnite allele and stepwise mutation models. Genetics Acyrthosiphon pisum. Entomologia experimentalis et appli- 140, 679–695. cata 51, 125–132. Goldstein, D. B. & Clark, A. G. (1995). Microsatellite Sokal, R. R. & Rohlf, F. J. (1995). Biometry: The Principles variation in North American populations of Drosophila and Practice of Statistics in Biological Research, 3rd edn. melanogaster. Nucleic Acids Research 23, 3882–3886. New York: W. H. Freeman. Hales, D. F. (1976). Juvenile hormone and aphid poly- Sunnucks, P. & Hales, D. F. (1996). Numerous transposed morphism. In: Phase and Caste Determination in Insects, sequences of mitochondrial cytochrome oxidases I–II in (ed. M. Lu$ scher), pp. 105–115. Oxford: Pergamon Press. aphids of the genus Sitobion (Hemiptera: Aphididae). Hales, D. F. (1989). The chromosomes of Schoutedenia Molecular Biology and Eolution 13,510–524. lutea (Homoptera, Aphidoidea, Greenideinae) with an Sunnucks, P., England, P. R., Taylor, A. C. & Hales, D. F. account of meiosis in the male. Chromosoma 98, 295–300. (1996). Microsatellite and chromosome evolution of Hales, D. F. & Lardner, R. M. (1988). Genetic evidence for parthenogenetic Sitobion aphids in Australia. Genetics the occurrence of a new species of Neophyllaphis Taka- 144, 747–756. hashi in Australia (Homoptera: Aphididae). Journal of White, M. J. D. (1973). The Chromosomes. London: Chap- the Australian Entomological Society 27,81–85. man & Hall.